CN114249909A - Micro-nano magnetic polymer microsphere with surface topological structure and preparation method thereof - Google Patents
Micro-nano magnetic polymer microsphere with surface topological structure and preparation method thereof Download PDFInfo
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Abstract
The invention discloses a micro-nano magnetic polymer microsphere, which comprises magnetic nanoparticles and a polymer coated with the magnetic nanoparticles, wherein the magnetic polymer microsphere has a topological surface and the particle size of 100 nm-100 mu m; the microspheres have no through-hole structure and have a specific surface area 2 to 100 times that of smooth spherical particles having the same particle size as the microspheres; after the micro-nano magnetic polymer microspheres are subjected to surface functionalization modification, the separation efficiency of a detected object can be improved.
Description
Technical Field
The invention relates to a novel micro-nano magnetic polymer microsphere, in particular to a polymer microsphere which has a complex surface topological structure and can be widely applied to the field of detection and separation of biological samples.
Background
The immunomagnetic beads are core materials for detection and separation in the life and health fields, and are key to realizing efficient separation and accurate detection of major disease markers such as nucleic acid, virus, bacteria and cells. For the isolated detection of circulating tumor cells, the immunomagnetic bead method is the only clinical diagnostic technique approved by the U.S. Food and Drug Administration (FDA) and the national drug administration (NMPA).
At present, the common methods for preparing the magnetic microspheres mainly comprise: (1) directly wrapping the polymer functionalized shell on the surface of the magnetic nano particles to form magnetic microspheres with core-shell structures; (2) mixing magnetic nanoparticles with polymer monomers, initiators and the like, and forming magnetic microspheres with the magnetic nanoparticles uniformly distributed inside through polymerization reaction; (3) firstly, preparing polymer microspheres, and then loading magnetic nanoparticles on the surfaces of the polymer microspheres. According to the current preparation method, in the process of forming the polymer microspheres, due to the limitation of surface tension, the prepared magnetic microspheres are mostly particles with smooth surfaces. The magnetic microspheres with smooth surfaces have limited specific surface areas, can be combined with fewer biofunctionalized binding agents, and cannot form good topological interaction with target micro-nano structures such as pseudo feet on the surfaces of tumor cells, so that the separation efficiency of targets is low. In the existing preparation method, the loading or coating of the magnetic particles and the formation of the microspheres are carried out step by step, so that the preparation process is complex, and the control means has limitation. In other methods, although the magnetic microspheres are formed by direct polymerization, the polymerization reaction is carried out on the premise of containing the magnetic nanoparticles, the control difficulty of the microspheres is increased, and the polymerization conditions are not easy to master.
Disclosure of Invention
The invention aims to develop a new generation of immunomagnetic beads with micro-nano topological structures on the surfaces, break through the limitation that the conventional immunomagnetic beads realize separation and detection only through chemical action, enhance the topological interaction between the immunomagnetic beads and a separation object through the micro-nano structures on the surfaces of the immunomagnetic beads, realize efficient biological separation of topological structure matching and molecular recognition cooperation, and provide technical support for subsequent clinical diagnosis, treatment and the like.
The invention provides a magnetic polymer microsphere with abundant surface topological structures, which can perform the synergistic action of topological matching and molecular recognition with a detected object (such as tumor cells and the like) after surface functional modification, thereby improving the separation efficiency of the detected object. And the method has simple preparation process and strong controllability. The size, the surface micro-nano morphology and the like of the obtained magnetic microspheres can be controlled by simply regulating and controlling the molecular weight, the concentration, the ratio of hydrophilic and hydrophobic chain segments, the reaction temperature and the like of the polymer, and the large-scale preparation is expected to be realized.
In order to achieve the purpose, the invention adopts the following technical scheme:
the micro-nano magnetic polymer microsphere is characterized by comprising magnetic nanoparticles and a polymer coating the magnetic nanoparticles, wherein the micro-nano magnetic polymer microsphere has a topological surface and the particle size is 100 nm-100 mu m;
the topological surface refers to that the polymer microsphere has a continuous and abundant rough and fluctuant surface topography structure and can form a topological structure matching with a detection object;
the microspheres have no through-hole structure and have a specific surface area 2 to 100 times that of smooth spherical particles having the same particle size as the microspheres;
its topological surface can be enumerated as: a fold structure, a short burr structure, a long burr structure, a flower-like structure, a sea urchin-like structure or a core-satellite-like structure with a small ball on the surface, wherein the burr structure is preferred;
in one preferred embodiment, the surface of the microsphere has more than 2 burr structures, preferably more than 5 burr structures, and more preferably more than 10 burr structures, the burr structures are columnar, tubular or rod-shaped protrusions on the surface of the microsphere, and the average length of burrs is more than 0.20 μm, preferably more than 0.5 μm; the average diameter of the cross section is more than 0.05 μm;
in one preferred embodiment, the polymer is a single type of polymer, not a blend of polymers.
Wherein the polymer is an amphiphilic block copolymer and has a hydrophobic chain segment and a hydrophilic chain segment; the molecular weight of the hydrophobic chain segment is 5 kDa-200 kDa; the molecular weight of the hydrophilic chain segment is 500 Da-100 kDa;
the hydrophobic segment includes, but is not limited to, one or more of polyglycolide/lactide copolymer (PLGA), polylactic acid (PLA), Polylactide (PGA), Polystyrene (PS), Polymethylmethacrylate (PMMA), Polydimethylsiloxane (PDMS), Polyisobutylene (PIB), Polycaprolactone (PCL);
the hydrophilic segment includes, but is not limited to, one or more of polyethylene glycol (PEG), poly-4-vinylpyridine (P4VP), polyacrylic acid (PAA), poly (methoxypolyethylene glycol methacrylate) (PPEGMA), poly N, N-dimethylacrylamide (PDMA);
in one preferred embodiment, the amphiphilic block copolymer is polyglycolide/lactide copolymer (PLGA) -polyethylene glycol (PEG) -polyglycolide/lactide copolymer (PLGA);
the magnetic nanoparticles are ferroferric oxide nanoparticles, and the magnetic nanoparticles account for 1-90% of the total weight of the micro-nano magnetic polymer microspheres;
the micro-nano magnetic polymer microsphere is prepared by the following method:
dissolving an amphiphilic block copolymer in an organic solvent, adding magnetic nanoparticles, emulsifying and dispersing the magnetic nanoparticles in an aqueous solution containing a surfactant, and volatilizing an oil-water emulsion solvent to obtain the micro-nano magnetic polymer microspheres;
wherein, the organic solvent includes but is not limited to one or more of dichloromethane, trichloromethane, 1, 2-dichloroethane, trichloroethane, dimethyl carbonate, dioxane, carbon tetrachloride, ethyl acetate, ethylene glycol dimethyl ether, benzene, toluene, xylene, tetrahydrofuran, N-dimethylformamide and acetone;
the concentration of the amphiphilic block copolymer in the organic solvent is less than or equal to the saturation concentration; more preferably 1g/L to 50g/L, and particularly preferably 10g/L to 30 g/L;
in one preferable scheme, the magnetic nanoparticles are ferroferric oxide nanoparticles, and the magnetic nanoparticles account for 1-90% of the total weight of the micro-nano magnetic polymer microspheres; more preferably 3% to 20%; particularly preferably 4% to 15%;
in one preferred embodiment, the specific conditions of the emulsification stirring are as follows: the temperature is 0-100 ℃, and the preferable temperature is 30-80 ℃; the stirring speed is 500-20000 rpm/min, and more preferably 1000-10000 rpm/min; the stirring time is 0-4 h, and the preferable time is 1-3 h;
in one preferred embodiment, the surfactants include, but are not limited to: polyvinyl alcohol (PVA), Sodium Dodecyl Sulfate (SDS), Cetyl Trimethyl Ammonium Bromide (CTAB), concentration is below saturation concentration, more preferably 1 g/L-50 g/L;
in one preferred scheme, the micro-nano magnetic polymer microspheres are prepared by the following method, as shown in fig. 1:
a) dissolving an amphiphilic block copolymer in an organic solvent, wherein the concentration of the amphiphilic block copolymer in the organic solvent is below the saturation concentration, adding magnetic nanoparticles, the weight percentage of which is 1-90% of the total weight of the micro-nano magnetic polymer microspheres, and stirring for dispersion;
b) and (b) adding the solution a) into an aqueous solution with the surfactant concentration below the saturated concentration, stirring to form an oil-in-water emulsion, and volatilizing the solvent to obtain the micro-nano magnetic polymer microspheres. Wherein the specific conditions of the emulsification and stirring are as follows: the temperature is 0-100 ℃, the stirring speed is 500-20000 rpm/min, and the stirring time is 0-4 h.
The invention further uses the micro-nano magnetic polymer to carry out surface functional modification, and the binding agent of which the surface is subjected to functional modification comprises but is not limited to one or more of DNA, polypeptide, aptamer and antibody, wherein the antibody is preferred.
In one preferred embodiment, the surface modification is carried out by the following method:
a) hydrolyzing the micro-nano magnetic polymer microspheres by using an acid solution with a certain concentration;
b) soaking the microspheres obtained in the step a) in a prepared MES solution of EDC/NHS;
c) grafting streptavidin SA on the microspheres obtained in the step b);
d) grafting the modified microspheres obtained in the step c) with biotinylated antibodies.
The invention further comprises the application of the functionalized modified micro-nano magnetic polymer, in particular to the application of the antibody modified micro-nano magnetic polymer in biological sample separation, preferably the application in tumor cell separation. The invention has the beneficial effects that:
according to the micro-nano magnetic polymer microsphere provided by the invention, the surface has rich micro-nano structures, and as the surface structure is rich, compared with the existing immunomagnetic beads, the surface area is increased, more functional modifiers can be combined, for example, more antibodies can be modified, meanwhile, a large number of surface nano structures can form good topological matching with a detection object, typically pseudo feet of tumor cells, and the separation efficiency of the detection object is improved.
The preparation method is characterized in that an emulsion solvent volatilization self-assembly method is preferably used in the preparation process, and the regulation and control of the surface structure of the micro-nano magnetic polymer microsphere can be realized only by simply changing the proportion of the block copolymer or the preparation temperature and the like, so that the obtained micro-nano magnetic polymer microsphere can be smooth in surface, can be in a short micro-nano structure, and can be in a long micro-nano structure. The size of the micro-nano magnetic polymer microsphere can be controlled by changing the concentration of the block copolymer.
Meanwhile, the method for preparing the micro-nano magnetic polymer microspheres does not need to adopt the traditional two-step complicated preparation process of loading/coating magnetic particles and forming the microspheres, only one-step simple emulsion solvent volatilization is needed, the micro-nano magnetic polymer microspheres with rich surface structures can be obtained, the yield is high, the synthesis process is simple, and the large-scale preparation is expected to be realized.
Drawings
FIG. 1 is a schematic diagram of micro-nano magnetic polymer microspheres prepared according to the invention
FIG. 2 is a scanning electron microscope photograph of the micro-nano magnetic polymer microsphere prepared in example 1
FIG. 3 is a scanning electron micrograph of the micro-nano magnetic polymer microspheres prepared in example 2
FIG. 4 is a scanning electron micrograph of the micro-nano magnetic polymer microspheres prepared in example 3
FIG. 5 is a scanning electron micrograph of the micro-nano magnetic polymer microspheres prepared in example 4
FIG. 6 is a scanning electron micrograph of the micro-nano magnetic polymer microspheres obtained in example 8
FIG. 7 scanning Electron micrograph of commercial magnetic beads of comparative example 1
FIG. 8 is a scanning electron micrograph of micro-nano magnetic polymer microspheres and commercial magnetic beads with surface topology structure of Experimental example 1 for tumor cell separation
FIG. 9 shows the separation efficiency of micro-nano magnetic polymer microspheres and commercial magnetic beads with surface topology structure for tumor cell separation in Experimental example 1
FIG. 10 shows the separation efficiency of micro-nano magnetic polymer microspheres and commercial magnetic beads with surface topology structure in Experimental example 2 for the separation of a small amount of tumor cells
Detailed Description
The technical solution of the present invention will be further described in detail with reference to specific embodiments. The following examples are merely illustrative and explanatory of the present invention and should not be construed as limiting the scope of the invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.
[ amphiphilic Block copolymer ]
The invention uses amphiphilic block copolymer to prepare polymer microsphere, uses the difference of hydrophilicity and hydrophobicity of different parts of the block, wherein the hydrophilic section tends to form the surface of the microsphere, and the lipophilic section forms the interior of the microsphere. Specific examples are:
the hydrophobic chain segment is selected from one or more of polyglycolide/lactide copolymer (PLGA), polylactic acid (PLA), Polylactide (PGA), Polystyrene (PS), polymethyl methacrylate (PMMA), Polydimethylsiloxane (PDMS), Polyisobutylene (PIB) and Polycaprolactone (PCL);
the hydrophilic chain segment is selected from one or more of polyethylene glycol (PEG), poly-4-vinylpyridine (P4VP), polyacrylic acid (PAA), poly (polyethylene glycol methyl ether methacrylate) (PPEGMA), and poly N, N-dimethylacrylamide (PDMA);
in order to obtain an ideal surface appearance, the molecular weight of the hydrophilic chain segment is controlled to be 500 Da-100 kDa, the molecular weight of the hydrophobic chain segment is controlled to be 5 kDa-200 kDa, and the burr structure on the surface of the microsphere can be changed by changing the proportion of the hydrophilic chain segment in the block copolymer. With the increase of the hydrophilic chain segment, the length of the burr is increased and then reduced, and the thickness of the burr is increased and then reduced.
The organic solvent is selected to dissolve the amphiphilic block copolymer, is immiscible with water, has a boiling point less than that of water, and is volatile at temperatures above room temperature and below 100 ℃. Specific examples thereof include: one or more of dichloromethane, trichloromethane, 1, 2-dichloroethane, trichloroethane, dimethyl carbonate, dioxane, carbon tetrachloride, ethyl acetate, ethylene glycol dimethyl ether, benzene, toluene, xylene, tetrahydrofuran, N, N-dimethylformamide and acetone.
[ magnetic nanoparticles ]
The magnetic nanoparticles endow the microspheres with magnetism for the subsequent separation of a target object, paramagnetic ferroferric oxide nanoparticles are preferred in the invention, and the paramagnetic ferroferric oxide nanoparticles have good dispersibility, high degree of agglomeration among particles, higher crystallinity and good dispersibility in a selected organic solvent, and the current common preparation method comprises the following steps: coprecipitation of different metal salts, high-temperature hydrothermal decomposition, a sol-gel method, polyol reduction, an electrochemical method and a microemulsion method. See, in particular, references: "Magnetic Iron Oxide Nanoparticles Synthesis, Stabilization, Vectorization, physical characteristics characterization, and Biological applications Chem. Rev.2008,108, 2064-2110"
[ micro-nano magnetic polymer microspheres ]
The micro-nano magnetic polymer microsphere obtained by the invention not only has magnetic nano particles in the interior of the microsphere, but also has more than 2 burr structures on the surface of the microsphere, preferably more than 5 burr structures, more preferably more than 10 burr structures, wherein the burr structures are columnar, tubular or rod-shaped protrusions on the surface of the microsphere, and the average length of burrs is more than 0.20 mu m, preferably more than 0.5 mu m; the average diameter of the cross section is more than 0.05 μm.
In the emulsion solvent evaporation self-assembly method preferably used in the invention, the temperature and stirring rate in the preparation process have influence on burr control except for changing the molecular weight and ratio of the hydrophilic and hydrophobic segments, and specifically:
the temperature of the preparation was varied: along with the rise of the temperature, the micro-nano burrs on the surface are gradually shortened.
Stirring speed: the stirring speed mainly influences the size of the microspheres and the volatilization time of the solvent, the larger the stirring speed is, the smaller the average size of the formed microspheres is, the higher the volatilization speed of the solvent is due to the large stirring speed, and the shorter burrs are due to the short stirring speed.
From this, it can be presumed that the control mechanism of the present invention is:
first, when a block copolymer is dissolved in an organic solvent, molecular chains are stretched in the organic solvent in a state where the polymer is completely dissolved, and randomly arranged. The organic solvent with the block copolymer dissolved is emulsified in the water solution containing the surfactant, and the oil drops are in a smooth spherical shape under the stabilizing action of the surfactant. As the low-boiling-point organic solvent is volatilized from emulsion droplets, the oil droplets are gradually shrunk, the concentration of the block copolymer is increased, the hydrophilic chain segments in the block copolymer tend to move towards an oil-water interface and are distributed outside, the hydrophobic chain segments are distributed inside, and more block copolymers are adsorbed on the oil/water interface. The hydrophilic and hydrophobic blocks and the surfactant are adsorbed on the interface cooperatively, so that the interface tension between the emulsion droplets and water is reduced, the interface of the emulsion droplets is not smooth any more, but becomes wavy, and the surface of each droplet extends out of a nano-sized coarse structure. When the hydrophobic polymer is selected to be a glassy polymer, the hydrophobic polymer becomes solid when the concentration is increased to a certain value, and a burr structure is formed.
Therefore, the emulsion solvent volatilization self-assembly method can be used for effectively and controllably adjusting the rough appearance of the burr structure and the surface of the microsphere.
[ functional load modification ]
The functional load modification of the micro-nano magnetic polymer microsphere is to introduce other functional molecules in a surface modification mode so that the micro-nano magnetic polymer microsphere has more functions.
The interaction between the microspheres and the separation target can be enhanced by surface functional modification. The surface of the binding agent is functionally modified, wherein the binding agent comprises one or more of DNA, polypeptide, aptamer and antibody, preferably antibody, the surface of the micro-nano magnetic polymer microsphere is modified by the antibody, besides the introduction of antigen-antibody interaction, the microsphere can form good topological matching with cells, and the micro-nano magnetic polymer microsphere and the cells can cooperate to further enhance the separation of tumor cells. The specific process is as follows:
a) hydrolyzing the micro-nano magnetic polymer microspheres by using an acid solution with a certain concentration;
b) soaking the microspheres obtained in the step a) in a prepared MES solution of EDC/NHS;
c) grafting streptavidin SA on the microspheres obtained in the step b);
d) grafting the modified microspheres obtained in the step c) with biotinylated antibodies.
[ tumor cell identification and isolation ]
Adding the microspheres with the modified antibodies on the surfaces and the cell suspension into a centrifugal tube, and carrying out mixed culture for a certain time and in a certain proportion. The antigen over-expressed on the surface of the tumor cell and the antibody modified on the surface of the microsphere can be specifically identified, and the antigen-antibody interaction is generated between the antigen and the antibody; meanwhile, the pseudopodia on the surface of the cell can be tightly wound and hold the nano structure on the surface of the microsphere, and topological interaction is generated between the micro-nano structure on the surface of the polymer microsphere and the pseudopodia on the surface of the cell. The molecular recognition of antigen-antibodies and topological matching between structures synergistically facilitate the interaction between cells and microspheres. This effect is stronger than the single molecule recognition between smooth magnetic beads and cells.
When the microspheres and the cells act for a period of time, the magnetic microspheres capturing the tumor cells are separated by using a magnetic device, and the cells are separated. The captured tumor cells were dispersed in a new medium, cell counting was performed, and the separation efficiency was calculated.
The following will explain the process of the present application by way of specific examples and comparative examples and fully evaluate the effects of the practice. Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.
Example 1:
the micro-nano magnetic polymer microsphere with the surface burr structure is realized by the following steps:
a) 0.3g of PLGA50k-PEG4k-PLGA50k(product of biological science and technology Co., Ltd., Dai, Shandong) was dissolved in 10mL of methylene chloride;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 40 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 40 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
The surface of the synthesized magnetic polymer microsphere is characterized by a burr structure by SEM. The results are shown in FIG. 2.
Example 2
The micro-nano magnetic polymer microsphere with the surface burr structure is realized by the following steps:
a) 0.15g of PLGA50k-PEG4k-PLGA50kDissolved in 10mL of dichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 40 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 40 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
The surface of the synthesized magnetic polymer microsphere is characterized by a burr structure by SEM. The results are shown in FIG. 3.
Example 3
The micro-nano magnetic polymer microsphere with the surface burr structure is realized by the following steps:
a) 0.15g of PLGA50k-PEG4k-PLGA50kDissolved in 10mL of dichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 30 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 30 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
The surface of the synthesized magnetic polymer microsphere is characterized by having a burr structure by SEM, and the result is shown in FIG. 4.
Example 4
The micro-nano magnetic polymer microsphere with the burr structure on the surface is realized by the following steps:
a) 0.15g of PLGA50k-PEG4k-PLGA50kDissolved in 10mL of dichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 60 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 60 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
The surface of the synthesized magnetic polymer microsphere is characterized by having a burr structure by SEM, and the result is shown in FIG. 5.
Example 5
The micro-nano magnetic polymer microsphere with the surface burr structure is realized by the following steps:
a) 0.15g of PLGA90k-PEG8k-PLGA90kDissolved in 10mL of dichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 40 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 40 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
Example 6
The micro-nano magnetic polymer microsphere with the surface burr structure is realized by the following steps:
a) 0.15g of PLGA50k-PEG4k-PLGA50kDissolved in 10mL of dichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 20g/L for 5min at 40 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 40 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
Example 7
The micro-nano magnetic polymer microsphere with the surface burr structure is realized by the following steps:
a) 0.15g of PLGA50k-PEG4k-PLGA50kDissolving in 10mL of trichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 40 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 40 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
Example 8:
the micro-nano magnetic polymer microsphere with the wrinkled structure on the surface is realized by the following steps:
a) dissolving 0.3g of polycaprolactone-polyethylene glycol copolymer PCL-PEG in 10mL of dichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 40 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 40 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
The surface of the synthesized magnetic polymer microsphere has a wrinkled structure by SEM characterization, and the result is shown in FIG. 6.
Example 9
The magnetic polymer microsphere with the surface burr structure is realized by the following steps:
a) 0.15g of PLGA70k-PEG4k-PLGA70kDissolved in 10mL of dichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 40 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 40 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
Comparative example 1:
comparative example 1 is a commercial magnetic bead: dynabeadsTM M-280Streptavidin, purchased from website https:// www.thermofisher.com/order/catalog/product/11205D #/11205D, and SEM-characterized magnetic polymer microspheres synthesized have a relatively smooth surface structure, and the results are shown in FIG. 7.
Comparative example 2
The magnetic polymer microsphere with smooth surface is realized by the following steps:
a) 0.15g of PLGA50k-PEG1k-PLGA50kDissolved in 10mL of dichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 40 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 40 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
Comparative example 3
The magnetic polymer microsphere with smooth surface is realized by the following steps:
a) 0.15g of PLGA50k-PEG4k-PLGA50kDissolved in 10mL of dichloromethane;
b) dispersing and stirring 200mL of polyvinyl alcohol aqueous solution with the concentration of 10g/L for 5min at 70 ℃ and 6000 rpm/min;
c) adding 10mg of ferroferric oxide nano particles into the solution in the step a), and stirring and dispersing;
d) slowly adding the solution of c) into the solution of b), and stirring for 2 hours at 70 ℃ and 6000 rpm/min;
e) and repeatedly washing the solution obtained by the reaction by deionized water for three times, and freeze-drying.
Experimental example 1:
the magnetic polymer microspheres obtained in example 1 and comparative example 1 are loaded with biotinylated epithelial cell adhesion molecule antibody biotin-anti-EpCAM, and tumor cells are identified and separated.
1. Weighing 10mg of magnetic polymer microspheres with a micro-nano structure, adding 2mol/L hydrochloric acid for hydrolysis, then adding 5mg/ml EDC, activating by 5mg/ml NHS, then modifying streptavidin SA, and finally grafting antibody biotin-anti-EpCAM. And dispersing the micro-nano magnetic polymer microspheres modified with the antibody in PBS for later use.
2. In commercial magnetic beads DynabeadsTMThe M-280Streptavidin surface grafted antibody biotin-anti-EpCAM is dispersed in PBS for later use.
3. Density 1051 ml of cell suspension/ml is mixed with two kinds of magnetic beads with proper amount respectively, the mixture is placed in an incubator for incubation, then a magnetic separator is used for separation, PBS is used for cleaning, cells captured by micro-nano magnetic polymer microspheres or commercial magnetic beads are suspended in PBS solution for cell counting, a cell counting plate is used for detecting the enriched cells, and the capture efficiency is calculated; and the interaction between the cells and the micro-nano magnetic polymer microspheres and magnetic beads is respectively observed through a scanning electron microscope, and the results are shown in fig. 8 and fig. 9.
As can be seen, a good topological interaction is formed between the magnetic polymer microsphere with the surface micro-nano structure and cells, and the cells extend out of the filamentous pseudo-feet and are wound on the surface of the microsphere. For commercial beads, cells do not form a good topological match with their surface, and fewer artifacts stick out. Therefore, from the results, the capture efficiency of the magnetic polymer microsphere with the micro-nano structure on the surface is higher than that of a commercial magnetic bead, so that the superiority of the magnetic polymer microsphere with the micro-nano structure in the invention is embodied.
Experimental example 2:
the magnetic polymer microspheres obtained in example 1 and comparative example 1 are loaded with antibody biotin-anti-EpCAM, and a small amount of tumor cells are identified and separated.
1. Weighing 10mg of magnetic polymer microspheres with a micro-nano structure, adding 2mol/L hydrochloric acid for hydrolysis, then adding 5mg/ml EDC, activating by 5mg/ml NHS, then modifying streptavidin SA, and finally grafting antibody biotin-anti-EpCAM. And dispersing the micro-nano magnetic polymer microspheres modified with the antibody in PBS for later use.
2. In commercial magnetic beads DynabeadsTMThe M-280Streptavidin surface grafted antibody biotin-anti-EpCAM is dispersed in PBS for later use.
3. Counting 10, 25, 50, 100, 200 and 500 cells by using a cell counter respectively, mixing the cells with two kinds of magnetic beads in proper amount respectively, placing the mixture in an incubator for incubation, separating the mixture by using a magnetic separator, washing the mixture by using PBS (phosphate buffer solution), suspending the cells captured by the magnetic beads in the PBS solution, counting the cells, detecting the enriched cells by using a cell counting plate, and calculating the capturing efficiency. The results are shown in FIG. 10.
From the results, the capture efficiency of the magnetic polymer microsphere with the micro-nano structure on the surface is higher than that of a commercial magnetic bead, so that the superiority of the magnetic polymer microsphere with the micro-nano structure in the invention is embodied.
Experimental example 3:
the magnetic polymer microspheres with different morphologies obtained in example 1, example 4 and comparative example 2 are loaded with antibody biotin-anti-EpCAM, and tumor cells are identified and separated.
1. Respectively weighing 10mg of the micro-nano magnetic polymer microspheres obtained in the embodiment 1, adding 2mol/L hydrochloric acid into the magnetic polymer microspheres with the shorter micro-nano structures on the surfaces obtained in the embodiment 4 and the magnetic polymer microspheres with smooth surfaces obtained in the comparative example 2 for hydrolysis, then adding 5mg/ml EDC, activating 5mg/ml NHS, then modifying streptavidin SA, and finally grafting antibody biotin-anti-EpCAM. And dispersing the micro-nano magnetic polymer microspheres modified with the antibody in PBS for later use.
2. Respectively taking 1 ml of cell suspension with the density of about 200/ml, mixing the cell suspension with an appropriate amount of three magnetic beads, placing the cell suspension in an incubator for incubation, then separating the cell suspension by using a magnetic separator, washing the cell suspension by using PBS (phosphate buffer solution), suspending the cells captured by the magnetic beads in the PBS solution, counting the cells, detecting the enriched cells by using a cell counting plate, and calculating the capturing efficiency. As shown in the following table:
TABLE 1 capture efficiency of magnetic polymer microspheres with different surface structures for tumor cells
From the results, the capture efficiency of the magnetic polymer microsphere with the longer micro-nano structure on the surface to cells is higher than that of the magnetic polymer microsphere with the shorter micro-nano structure and higher than that of the magnetic polymer microsphere with the smooth surface, so that the superiority of the magnetic polymer microsphere with the topological structure on the surface in tumor cell separation is embodied.
Claims (25)
1. The micro-nano magnetic polymer microsphere is characterized by comprising magnetic nanoparticles and a polymer coating the magnetic nanoparticles, wherein the micro-nano magnetic polymer microsphere has a topological surface and the particle size is 100 nm-100 mu m;
the topological surface means that the polymer microsphere has a continuous and abundant rough and fluctuant surface morphology structure and can form a topological structure matching with a detection object.
2. The micro-nano magnetic polymer microsphere according to claim 1, which has no through-hole structure and has a specific surface area 2 to 100 times that of smooth spherical particles having the same particle size as the microsphere.
3. The micro-nano magnetic polymer microsphere according to any one of claims 1 to 2, wherein the topological surface is: a fold structure, a burr structure, a flower-like structure, a sea urchin structure or a core-satellite-like structure with a small ball on the surface, wherein the burr structure is preferred.
4. The micro-nano magnetic polymer microsphere according to any one of claims 1 to 3, wherein the surface of the micro-nano magnetic polymer microsphere has more than 2 burr structures, preferably more than 5 burr structures, more preferably more than 10 burr structures, the burr structures are columnar, tubular or rod-shaped protrusions on the surface of the microsphere, and the average length of burrs is more than 0.20 μm, preferably more than 0.5 μm; the average diameter of the cross section is more than 0.05 μm.
5. The micro-nano magnetic polymer microsphere of claim 1, wherein the polymer is a single polymer, not a blend of multiple polymers.
6. The micro-nano magnetic polymer microsphere according to claim 1, wherein the polymer is an amphiphilic block copolymer and has a hydrophobic chain segment and a hydrophilic chain segment;
the amphiphilic block copolymer is a diblock copolymer or a triblock copolymer or a copolymer of more than three blocks.
7. The micro-nano magnetic polymer microsphere of claim 6, wherein the molecular weight of the hydrophobic segment of the block copolymer is 5-200 kDa; the molecular weight of the hydrophilic chain segment is 500 Da-100 kDa.
8. The micro-nano magnetic polymer microsphere according to any one of claims 6 to 7, wherein the hydrophobic segment comprises one or more of polyglycolide/lactide copolymer (PLGA), polylactic acid (PLA), Polylactide (PGA), Polystyrene (PS), polymethyl methacrylate (PMMA), Polydimethylsiloxane (PDMS), Polyisobutylene (PIB), Polycaprolactone (PCL).
9. The micro-nano magnetic polymer microsphere according to any one of claims 6 to 8, wherein the hydrophilic segment comprises one or more of polyethylene glycol (PEG), poly-4-vinylpyridine (P4VP), polyacrylic acid (PAA), poly (polyethylene glycol methyl ether methacrylate) (PPEGMA), poly N, N-dimethylacrylamide (PDMA).
10. The micro-nano magnetic polymer microsphere according to any one of claims 6 to 9, wherein the amphiphilic block copolymer is polyglycolide/lactide copolymer (PLGA) -polyethylene glycol (PEG) -polyglycolide/lactide copolymer (PLGA).
11. The micro-nano magnetic polymer microsphere according to any one of claims 1 to 10, wherein the magnetic nanoparticles are ferroferric oxide nanoparticles, and the magnetic nanoparticles are 1 to 90 wt% based on the total weight of the micro-nano magnetic polymer microsphere.
12. The micro-nano magnetic polymer microsphere according to any one of claims 1 to 11, prepared by emulsion solvent volatilization self-assembly; the preparation method specifically comprises the steps of dissolving the amphiphilic block copolymer in an organic solvent, adding the magnetic nanoparticles, emulsifying and dispersing the magnetic nanoparticles in an aqueous solution containing a surfactant, and volatilizing an oil-water emulsion solvent to obtain the micro-nano magnetic polymer microspheres.
13. The micro-nano magnetic polymer microsphere of claim 12, wherein the organic solvent comprises one or more of but not limited to dichloromethane, chloroform, 1, 2-dichloroethane, trichloroethane, dimethyl carbonate, dioxane, carbon tetrachloride, ethyl acetate, ethylene glycol dimethyl ether, benzene, toluene, xylene, tetrahydrofuran, N-dimethylformamide, and acetone.
14. The micro-nano magnetic polymer microsphere according to claims 12 to 13, wherein the concentration of the amphiphilic block copolymer in the organic solvent is below the saturation concentration.
15. The micro-nano magnetic polymer microsphere according to any one of claims 12 to 14, wherein the specific conditions of emulsification and stirring are as follows: the temperature is 0-100 ℃, the stirring speed is 500-20000 rpm/min, and the stirring time is 0-4 h.
16. The micro-nano magnetic polymer microsphere according to any one of claims 12 to 15, wherein the surfactant includes but is not limited to: polyvinyl alcohol (PVA), Sodium Dodecyl Sulfate (SDS), Cetyl Trimethyl Ammonium Bromide (CTAB), concentration is below saturation concentration.
17. The preparation method of the micro-nano magnetic polymer microsphere according to any one of claims 1 to 16, wherein the micro-nano magnetic polymer microsphere comprises the following steps:
dissolving the amphiphilic block copolymer in an organic solvent, adding magnetic nanoparticles, emulsifying and dispersing the magnetic nanoparticles in an aqueous solution containing a surfactant, and volatilizing an oil-water emulsion solvent to obtain the micro-nano magnetic polymer microspheres.
18. The method according to claim 17, wherein the organic solvent is selected from one or more of dichloromethane, chloroform, 1, 2-dichloroethane, trichloroethane, dimethyl carbonate, dioxane, carbon tetrachloride, ethyl acetate, ethylene glycol dimethyl ether, benzene, toluene, xylene, tetrahydrofuran, N-dimethylformamide, and acetone.
19. The production method according to claims 17 to 18, wherein the concentration of the amphiphilic block copolymer in the organic solvent is below a saturation concentration.
20. The production method according to any one of claims 17 to 19, wherein the specific conditions of the emulsification stirring are: the temperature is 0-100 ℃, the stirring speed is 500-20000 rpm/min, and the stirring time is 0-4 h.
21. The micro-nano magnetic polymer microsphere according to any one of claims 17 to 20, wherein the surfactant includes but is not limited to: polyvinyl alcohol (PVA), Sodium Dodecyl Sulfate (SDS), Cetyl Trimethyl Ammonium Bromide (CTAB), concentration is below saturation concentration.
22. The method of claim 17, further comprising:
a) dissolving an amphiphilic block copolymer in an organic solvent, wherein the concentration of the amphiphilic block copolymer is below saturated concentration, adding magnetic nanoparticles, the weight percentage of the magnetic nanoparticles is 1-90% based on the total weight of the micro-nano magnetic polymer microspheres, and stirring and dispersing;
b) adding the solution a) into an aqueous solution with the surfactant concentration below the saturation concentration, stirring to form an oil-in-water emulsion, and further volatilizing the solvent to obtain the micro-nano magnetic polymer microsphere, wherein the specific conditions of emulsification and stirring are as follows: the temperature is 0-100 ℃, the stirring speed is 500-20000 rpm/min, and the stirring time is 0-4 h.
23. A functionalized micro-nano magnetic polymer microsphere, which is characterized in that the surface of the micro-nano magnetic polymer microsphere of claims 1 to 16 is functionalized and modified by a binding agent, wherein the binding agent comprises one or more of but not limited to DNA, polypeptide, aptamer and antibody.
24. The surface-functionalized micro-nano magnetic polymer microsphere according to claim 23, wherein preferably the binding agent is an antibody, and the modification method comprises the following steps:
a) hydrolyzing the micro-nano magnetic polymer microspheres by using an acid solution with a certain concentration;
b) soaking the microspheres obtained in the step a) in a prepared 2-morpholine ethanesulfonic acid MES solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride/N-hydroxysuccinimide EDC/NHS;
c) grafting streptavidin SA on the microspheres obtained in the step b);
d) grafting the modified microspheres obtained in the step c) with biotinylated antibodies to obtain the modified microspheres modified by the antibodies.
25. Use of the functionalized micro-nano magnetic polymer microspheres according to claims 23-24 for biological sample separation, preferably for tumor cell separation.
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